This application claims benefit of Norwegian Patent Application No. 2000 0855, filed Feb. 21, 2000.
The present invention relates to a conjugator system comprising liposomes with ionophores and with chelator located inside of the liposomes, wherein the liposomes are stably labeled with heavy radionuclides emitting a particles. The present invention further relates to a method to prepare the conjugator system and use of the system, as well as a kit for preparing the conjugator system.
Biomedical applications of radionuclides in anticancer therapy have so far focused on the use of cationic or anionic species, e.g., [131I]iodide against thyroid cancer and 89Sr for palliation of pain from skeletal cancer metastases, and the use of mostly beta-emitting radionuclides attached to monoclonal antibodies (DeVita et al., 1996).
The use of targeted radionuclide therapy against cancer rest upon the ability to find ways to attach radionuclides to tumor specific carrier compounds (Gaze, 1996). Today, some of the radionuclides with useful radiation characteristics cannot be used in tumor targeting because of the problem of providing a chemically stable link between the radionuclide and the carrier compound. Now carrier systems may, however, broaden both the use of radioisotopes, as well as the arsenal of radionuclides considered useful for therapy (Gaze, 1996).
Liposomes, with or without receptor affinic groups attached to the surface, have been evaluated for drug delivery, and is currently used clinically for the delivery of chemotherapeutics in some cancer forms. Recently, developments in liposome research have led to new versions with a pharmacokinetics which could make these compounds useful as carriers for radionuclide for internal radiotherapy against cancer (Gabizon, 1995). These recent developments in the formulation and manufacturing of liposomes have resulted in small vesicles of less than 100 nm with prolonged circulation time, as the size of the liposomes can be better confined to small diameters by using extrusion through membranes. Furthermore, the introduction of poly ethylene glycol (PEG) grafted liposomes has reduced the interference from plasma proteins, and thus reduced the recognition and clearance affected by the macrophages of the reticuloendothelial system (Maruyama, et al., 1997). Increased levels of tumor uptake due to sustained blood concentration have thereby been achieved. Even further tumor uptake has boon achieved by conjugating molecules with receptor affinity, e.g., monoclonal antibodies or folate, to the surface of the liposomes. In addition, several studies have indicated the advantage of applying PEG as a linker between the lipsome and the targeting ligand, since this also can improve the receptor accessibility (e.g. Maruyama et al, 1997; Gabison et al, 1999; Lee et al, 1994).
Liposomes have previously been studied as carriers for radioisotopes (Goins et al, 1998; Turner et al, 1988). Pikul, et al (1987) reported a study based on 212Pb-dextran incorporated passively (i.e., the 212Pb-dextran was added during the generation of the liposome, and a fraction of the 212Pb-dextran was incorporated together with the aqueous phase representing the interior of the liposome). The authors did not suggest that these liposomes were suitable for cancer therapy, but was using it primarily for studying intracellular cell killing in vitro with radioisotope. No data of the fate of 212Bi generated from the 212Pb decay was presented, and the size of the liposomes were in the order of 350-500 nm which is very large compared to the size (approx.100 nm) currently considered optimal for in vivo tumor therapy (Forssen, 1997). Also, the liposomes did not contain PEG in the membrane.
Ogihar-Umeda et al. (1996) used liposomes as carrier for the gamma emitting radionuclides 67Ga, 111In and 99mTc, and suggested the use of radiolabeled liposomes for imaging.
In a theoretical study, Kostarelos et al. (1999) suggested the use of liposomes labeled with the potentially therapeutically radionucides 131I, 67Cu, 138Re and 211At, but chemical procedures for the preparation of the radiolabeled liposomes were not suggested.
EP386 146 B1 describes a composition and method of use for liposome encapsulated compounds for neutron capture tumor therapy. However, these liposomes were loaded with stabel elements (e.g. boron), that become radioactive only after activation, and the liposomes contain neither ionophores nor chelator.
Utkhede et al., (1994) describes liposomes loaded with 90Y and the chelator DTPA, which is a different chelator compared to the chelators described in the present invention. Furthermore, retention of mother/daughternuclide(s) is not described and in addition, 90Y is not a heavy element as the elements described in the present application.
Achieving sufficiently stable radiolabeling of carriers with heavy alpha-emitting radionuclides usually requires specific chemical procedures tailored to suit the chemistry of each element, and such methods are not known. Procedures used to radiolabel e.g. Ga, In, Te, Cu or Re cannot be expected to be compatible with heavy radionuclide (atomic weight over 150) cationic alpha emitters like e.g. 212/213Bi, 212Pb, 223/224Ra, 227Th or 225Ac.
It is therefore an object of the present invention to provide a radionuclide-liposome conjugator system, with or without receptor affinic groups, that (1) encapsulates chelator and heavy radionuclide(s) that emit alpha particle radiation, (2) can retain daughter nuclide(s) when the mother nuclide(s) is incorporated in the liposome and (3) can be prepared by an active incorporation procedure useful for a panel of radionuclides, as well as use of the system and a kit for preparing the system These objects have been obtained by the present invention, characterized by the enclosed claims.
The present invention relates to a conjugator system comprising liposomes with ionophores, i.e., a metal extracting agent for liposomes, and with chelator and heavy radionuclide(s) (atomic weight over 150) located inside the liposome. The liposomes are prepared using active incorporation of the radionuclide, i.e., via ionophores, and prepared according to procedures yielding liposomes of size of typically 100 nm. The resulting product shows good chemical stability over several days, and may also retain daughter nuclide(s) inside the liposome, e.g. from the transformation of 212Pb to 212Bi. The liposomes can be prepared with or without modifying groups like polyalkylene oxides, e.g., PEG, attached to the membrane. Herein we also describe a method to link such radiolabeled liposomes to tumor seeking proteins, such as e.g. folate conjugated monoclonal antibodies.